This competitive renewal stems from our finding that the nonsense-mediated mRNA decay (NMD) factor UPF1 has multiple functions in mammalian cells. Our result using a stringent yeast-two-hybrid assay that human UPF1 interacts with human Staufen (STAU)1 led to our discovering a pathway related to NMD that we named STAU1-mediated mRNA decay (SMD). We have demonstrated that STAU1 recruits UPF1 to the 3'- untranslated region (3'-UTR) of particular mRNAs so as to elicit mRNA decay when translation terminates normally. Unlike NMD, which functions primarily as a quality control mechanism to eliminate mRNAs that prematurely terminate translation during a pioneer round of translation, SMD appears to conditionally down- regulate the expression of genes whose mRNAs bind STAU1 within their 3'-UTRs during both pioneer and subsequent rounds of translation. AIM 1 will continue to examine the mechanism of SMD by characterizing the factors involved, the role for STAU1 arginine methylation, the cis-acting mRNA sequences that bind STAU1, and the proteins other than STAU1 that bind SMD targets. In related studies, we have found that STAU1 can down-regulate the expression of particular genes in a way that is insensitive to UPF1 siRNA and, thus, apparently distinct from SMD. To follow up these unpublished results, AIM 2 will define the one or more STAU1-mediated pathways of post-transcriptional gene regulation that are apparently distinct from SMD. We will focus on pathways that affect mRNA metabolism, which is our forti. We have additionally discovered that hyper-phosphorylated UPF1 functions during NMD to repress translation by binding to eIF3 in a way that prevents joining of the 60S ribosomal subunit to the 40S ribosomal subunit that constitutes the preinitiation complex at the 5'-end of an NMD target. AIM 3 will further characterize the interaction of hyper- phosphorylated UPF1 with eIF3 as a means to better understand the mechanism of translational repression. The proposed experiments logically extend our long-time studies of RNA metabolism in mammalian cells and are expected to lend important insights into aspects of SMD, one or more new STAU1-dependent pathways of post-transcriptional gene control, and translational repression during NMD. PUBLIC HEALTH RELEVANCE: Proper control of human gene expression requires the two mRNA decay pathways that are at the heart of this competitive renewal. Both pathways were uncovered by this P.I. Staufen1- mediated mRNA decay serves to conditionally down-regulate genes encoding mRNAs that bind Staufen1 in their 3'-untranslated regions. Nonsense-mediated mRNA decay (NMD) eliminates the expression of genes encoding mRNAs that harbor premature termination codons. By so doing, NMD protects many heterozygous carriers of disease-associated alleles from manifesting a dominantly inherited form of the disease. NMD also contributes to proper gene expression in many ways. As an example, NMD provides a means by which many RNA binding proteins negatively regulate the genes from which they derived so that an appropriate level of gene expression can be achieved.